The goal of these studies is to understand mechanisms of cross- communication between the major transmembrane signal pathways (i.e., inositol phosphates, cAMP) in vascular smooth muscle cells that are key to the regulation of vascular tone. We have observed a pronounced effect of the pressor peptide, angiotensin II, to synergistically enhance the action of dilator agonists (isoproterenol, PGI-2, adenosine) to stimulate cAMP formation in cultured vascular smooth muscle cells. The objectives of this proposal are to determine the molecular basis for this observed synergism and to evaluate the significance of such signal cross-talk to the modulation of vascular tone. A working hypothesis is that angiotensin II enhancement of hormone-induced cAMP formation results from the action of angiotensin II to elevate intracellular free Ca2+ which acts synergistically with the activated stimulatory guanine nucleotide regulatory protein, G2, to stimulate a calmodulin-sensitive form of adenylyl cyclase in the membrane of vascular smooth muscle cells. It is also proposed that calcium-mobilizing constrictor agonists which enhance cAMP stimulation in vascular smooth muscle cells will sensitize blood vessels to the relaxant effects of dilator agents that act through adenylyl cyclase activation. To test these hypotheses, studies will be conducted with isolated aortic rings and with particulate membranes and partially purified adenylyl cyclases isolated from a homogeneous population of cultured vascular smooth muscle cells. Experiments are proposed: i) to determine the effects of calcium-mobilizing constrictor agonists on the relaxation of aortic rings by dilator agents (isoproterenol, prostacyclin) that act through cAMP in comparison to dilator agents (nitroprusside, ANF) that act through alternative mechanisms; 2) to characterize Ca2+-calmodulin regulation of basal and stimulated adenylyl cyclase in particulate membranes and in a partially- purified enzyme preparation where G2 remains effectively coupled to the catalytic unit of adenylyl cyclase; and 3) to examine the influence of activated G proteins (G5, Gi,Go) and the beta-gamma subunit complex of these proteins on the regulation of different adenylyl cyclase isoforms solubilized from vascular smooth muscle cells and separated using calmodulin-sepharose affinity chromatography (preliminary studies support the feasibility of this approach). These studies should provide new information on the cellular processes that participate in hormonal control of vascular tone and have the potential to substantially broaden our perspective on the possible mechanisms that may underlie hypertension and other forms of vascular disease.